Lymphocytes express a vast repertoire of antigen receptors, but only one species of receptor is allowed per cell in order for them to respond specifically to pathogens. The diversity of the receptors is generated by V(D)J rearrangement and, in immunoglobulin (Ig) genes, further altered by post- rearrangement somatic hypermutation (SHM) and class switch recombination. All these processes involve DNA strand breakage. It is important to understand the regulation of these pathways, which are part of normal B lymphocyte development. Missteps in the process have been shown to influence or directly initiate oncogenesis. The proposed research is to be done in sharks, representatives of the earliest vertebrates with an adaptive immune system based on V(D)J recombination. Whereas in mouse and human systems there are 3 Ig loci with hundreds of rearranging genes, sharks have a unique Ig gene organization of up to 200 loci, each one containing very few (2-4) recombining elements. This organizational disparity between cartilaginous fishes and tetrapods affords a unique opportunity to gain insight into the complex process governing Ig gene expression by elucidating shared and divergent regulatory mechanisms. Specific Aims 1 and 3 focus on patterns of activity among the many miniloci (27 IgH, 51 IgL) during V(D)J rearrangement. The Ig genes in individual B cells will be examined at the genomic and mRNA level in order to understand interplaying factors that bring about the Ig receptor ultimately expressed. Because preliminary results indicate that more Ig genes appear to be activated than strictly required to make a single functional receptor, experiments are proposed to 1) find if the commonly accepted linkage of rearrangement with transcriptional activity is decoupled in this instance, as part of the regulatory process, and 2) establish if DNA recombination is additionally employed to incapacitate unwanted (e.g. auto-reactive) Ig genes. Specific Aim 2 investigates recombination events in shark B cells initiated by activation-induced cytidine deaminase (AID). AID causes SHM and insertions/deletions not only at the genes encoding the Ig receptor but multiple other Ig sites, so that nicks and double-strand breaks are introduced throughout the genome after lymphocyte activation. We discovered that VDJ from one IgH can be expressed with the C region from another IgH, occurring sometimes after unequal crossing-over between the spatially distant genes. We propose isolating the recombination products that do not encode the receptor, including non- expressed reciprocal products of crossing-over events, in order to clarify the conditions under which partner genes are targeted for recombination. Understanding normal processes in shark B cells that lead to genetic exchange between non-allelic genes is fundamental to understanding pathogenesis of disease states such as autoimmunity and predisposal to lymphoid tumors by aberrant translocations involving Ig genes.